Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.     

Mutation screening using automated bidirectional dideoxy fingerprinting

The need continues to grow for mutation identification in genetic disease in both research and clinical settings. We have developed a rapid nonradioactive bidirectional dideoxy fingerprint mutation screening procedure that is performed using an automated DNA analyzer. This technique features standardized primers and easily interpreted results from separate, but simultaneously collected, images for coding and noncoding strands. Another advantage is simplified mutation verification by sequencing using the same amplified DNA templates and also application to large multi-exon genes. We demonstrate the efficiency and reproducibility of the method in which we screen a DNA fragment encompassing exon 5 of the PTCH gene (in which mutations cause Gorlin Syndrome) in a panel of 22 patients.     

Rapid automated detergent screening for the solubilization and purification of membrane proteins and complexes

Membrane proteins constitute about one third of proteins encoded by all genomes, but only a small percentage have their structures deposited in the Protein Data Bank. One bottleneck in the pipeline from expression to structure determination is the identification of detergents that maintain the protein in a soluble, stable, and active state. Here, we describe a small‐scale automated procedure to easily and rapidly screen detergents for the solubilization and purification of membrane proteins, to perform detergent exchange, or to identify conditions preserving protein interactions in complexes. Hundreds of conditions can be tested in a few hours to select detergents that keep proteins folded and nonaggregated, from single membrane preparations of cells overexpressing the protein(s) of interest. Thirty‐one prokaryotic, eukaryotic, and viral membrane proteins were analyzed by our small‐scale procedure to identify the best‐associated detergents. Examples of results obtained with a bitopic and multitopic membrane proteins and membrane protein complexes are presented in more detail. DDM, DM, DMNG, TritonX‐100, LAPAO, and Fos‐12 appeared effective for successful membrane solubilization and protein purification of most selected targets. Eukaryotic proteins are in general more difficult to extract and purify from Escherichia coli membranes than prokaryotic proteins. The protocol has been developed for His‐tagged proteins, but can readily be adapted to other affinity tags by adjusting the chromatography resin and the buffer composition.     

An automated small-scale protein expression and purification screening provides beneficial information for protein production

One of the first key steps in structural genomics is high-throughput expression and rapid screening to select highly soluble proteins, the preferred candidates for crystal production. Here we describe the methodology used at the Berkeley Structural Genomics Center (BSGC) for automated parallel expression and small-scale purification of fusion proteins using a 96-well format. Our robotic method includes cell lysis, soluble fraction separation and purification with affinity resins. For detection of His-tagged proteins in the soluble fractions and after affinity resin elution, a dot-blot procedure with an anti-His-antibody is used. The expression level and molecular mass of recombinant proteins are checked by SDS-PAGE. With this approach, we are able to obtain beneficial information to be used for large-scale protein expression and purification.     

Comparison of relative mRNA quantification models and the impact of RNA integrity in quantitative real-time RT-PCR

Relative quantification in quantitative real-time RT-PCR is increasingly used to quantify gene expression changes. In general, two different relative mRNA quantification models exist: the delta-delta Ct and the efficiency-corrected Ct model. Both models have their advantages and disadvantages in terms of simplification on the one hand and efficiency correction on the other. The particular problem of RNA integrity and its effect on relative quantification in qRT-PCR performance was tested in different bovine tissues and cell lines (n = 11). Therefore different artificial and standardized RNA degradation levels were used. Currently fully automated capillary electrophoresis systems have become the new standard in RNA quality assessment. RNA quality was rated according the RNA integrity number (RIN). Furthermore, the effect of different length of amplified products and RNA integrity on expression analyses was investigated. We found significant impact of RNA integrity on relative expression results, mainly on cycle threshold (Ct) values and a minor effect on PCR efficiency. To minimize the interference of RNA integrity on relative quantification models, we can recommend to normalize gene expression by an internal reference gene and to perform an efficiency correction. Results demonstrate that innovative new quantification methods and normalization models can improve future mRNA quantification.     

RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation

Recent studies have uncovered an unanticipated diversity of noncoding RNAs (ncRNAs), although these studies provide limited insight into their biological significance. Numerous general methods for identification and characterization of protein interactions have been developed, but similar approaches for characterizing cellular ncRNA interactions are lacking. Here we describe RNA Affinity in Tandem (RAT), an original, entirely RNA tag-based method for affinity purification of endogenously assembled RNP complexes. We demonstrate the general utility of RAT by isolating RNPs assembled in vivo on ncRNAs transcribed by RNA polymerase II or III. Using RAT in conjunction with protein identification by mass spectrometry and protein-RNA interaction assays, we define and characterize previously unanticipated protein subunits of endogenously assembled human 7SK RNPs. We show that 7SK RNA resides in a mixed population of RNPs with different protein compositions and responses to cellular stress. Depletion of a newly identified 7SK RNP component, hnRNP K, alters the partitioning of 7SK RNA among distinct RNPs. Our results establish the utility of a generalizable RNA-based RNP affinity purification method and provide insight into 7SK RNP dynamics.     

High-throughput protein purification using an automated set-up for high-yield affinity chromatography

One of the key steps in high-throughput protein production is protein purification. A newly developed high-yield protein purification and isolation method for laboratory scale use is presented. This procedure allows fully automated purification of up to 60 cell lysates with milligram yields of pure recombinant protein in 18.5h. The method is based on affinity chromatography and has been set up on an instrument that utilizes positive pressure for liquid transfer through columns. A protocol is presented that includes all steps of equilibration of the chromatography resin, load of sample, wash, and elution without any manual handling steps. In contrast to most existing high-throughput protein purification procedures, positive pressure is used for liquid transfer rather than vacuum. Positive pressure and individual pumps for each liquid channel contribute to controlled flow rates and eliminate the risk of introducing air in the chromatography resin and therefore ensure stable chromatography conditions. The procedure is highly reproducible and allows for high protein yield and purity.